Assembly and analysis of Sinipercidae fish sex chromosomes reveals that a supergene drives sex chromosome origin and turnover

Abstract

The sex chromosomes of fish are usually homomorphic and recently derived, making them an ideal model to understand the origin and evolution of sex chromosomes in vertebrates. Here, combined Pacbio, Hi-C, and Illumina sequencing of the male mandarin fish (Siniperca chuatsi) genome enabled the assembly of a high-quality chromosome-level genome, including highly resolved X and Y chromosome assemblies. Genome wide association study (GWAS) and coverage information analysis revealed a 2.0 Mbp sex determining region (SDR). A Y-specific duplication of the anti-Müllerian hormone (amh) gene, amhy, was identified that encoded a truncated AMH protein. Loss of function and overexpression experiments demonstrated that amhy may act as a male sex-determining gene (SDG). GWAS led to the identification of a common sex chromosome (Chr24) and SDG (amhy) in S. scherzeri, while another sex chromosome (Chr11) was identified in Coreoperca whiteheadi. Interestingly, completely conserved protein coding sequences of amhy were commonly identified in male S. chuatsi, S. scherzeri, and S. knerii. These results support a classic model for the origin and evolution of early sex chromosomes and suggest that amhy is a super SDG that can drive the origin and turnover of sex chromosome in vertebrates.

Keywords

Mandarin fish / Sex chromosome assembly / Amhy / Sex-determining gene / Origin and turnover of sex chromosome / Biological Sciences / Genetics

References

[1] AbbottJK, NordénAK, HanssonB. Sex chromosome evolution: historical insights and future perspectives. Proc Roy Soc B-Biol Sci, 2017, 284: 20162806.

[2] BachtrogD. Y-chromosome evolution: emerging insights into processes of Y-chromosome degeneration. Nat Rev Genet, 2013, 14: 113-124.

[3] BachtrogD, HomE, WongKM, MasideX, de JongP. Genomic degradation of a young Y chromosome in Drosophila miranda. Genom Biol, 2008, 9: R30.

[4] BaileyJA, CarrelL, ChakravartiA, EichlerEE. Molecular evidence for a relationship between LINE-1elements and X chromosome inactivation: the Lyon repeat hypothesis. Proc Natl Acad Sci USA, 2000, 97: 6634-6639.

[5] BellottDW, HughesJF, SkaletskyH, BrownLG, PyntikovaT, ChoTJ, KoutsevaN, ZaghlulS, GravesT, RockS, et al. . Mammalian Y chromosomes retain widely expressed dosage-sensitive regulators. Nature, 2014, 508: 494-499.

[6] BellottDW, SkaletskyH, ChoTJ, BrownL, LockeD, ChenN, et al. . Avian W and mammalian Y chromosomes convergently retained dosage-sensitive regulators. Nat Genet, 2017, 49: 387-394.

[7] ChangCH, LarracuenteAM. Heterochromatin-enriched assemblies reveal the sequence and organization of the Drosophila melanogaster Y chromosome. Genetics, 2018, 211: 333-348.

[8] ChenJ, FanZ, TanD, JiangD, WangD. A review of genetic advances related to sex control and manipulation in tilapia. J World Aquac Soc, 2018, 49: 277-291.

[9] DingM, LiXY, ZhuZX, ChenJH, LuM, ShiQ, WangY, LiZ, ZhaoX, WangT, et al. . Genomic anatomy of male-specific microchromosomes in a gynogenetic fish. PLoS Genet, 2021, 17: e1009760.

[10] DuanW, GaoFX, ChenZW, GaoY, GuiJF, ZhaoZ, ShiY. A sex-linked SNP mutation in amhr2 is responsible for male differentiation in obscure puffer (Takifugu obscurus). Mol Biol Rep, 2021, 48: 6035-6046.

[11] EinfeldtAL, KessT, MessmerA, DuffyS, WringeBF, FisherJ, HeyerCD, BradburyLR, RuzzanteDE, BentzenP. Chromosome level reference of Atlantic halibut Hippoglossus hippoglossus provides insight into the evolution of sexual determination systems. Mol Ecol Res, 2021, 21: 1686-1696.

[12] El TaherA, RoncoF, MatschinerM, SalzburgerW, BöhneA. Dynamics of sex chromosome evolution in a rapid radiation of cichlid fishes. Sci Adv, 2021, 7: eabe8215.

[13] Ferchaud AL, Mérot C, Normandeau E, Ragoussis J, Babin C, Djambazian H, Bérubé P, Audet C, Treble M, Walkusz, W, et al. Chromosome-level assembly reveals a putative Y-autosomal fusion in the sex determination system of the Greenland Halibut (Reinhardtius hippoglossoides). G3. 2022;12:jkab376. https://doi.org/10.1093/g3journal/jkab376.

[14] FeronR, ZahmM, CabauC, KloppC, RoquesC, BouchezO, EchéC, ValièreS, DonnadieuC, HaffrayP, et al. . Characterization of a Y-specific duplication/insertion of the anti-Mullerian hormone type II receptor gene based on a chromosome-scale genome assembly of yellow perch, Perca flavescens. Mol Ecol Res, 2020, 20: 531-543.

[15] GongG, XiongY, XiaoS, LiXY, HuangP, LiaoQ, HanQ, LinQ, DanC, ZhouL, et al. . Origin and chromatin remodeling of young X/Y sex chromosomes in catfish with sexual plasticity. Nat Sci Rev, 2023, 10: nwac239.

[16] GravesMJA, PeichelCL. Are homologies in vertebrate sex determination due to shared ancestry or to limited options?. Genom Biol, 2010, 11: 1-12.

[17] GvozdevVA, KoganGL, UsakinLA. The Y chromosome as a target for acquired and amplified genetic material in evolution. BioEssays, 2005, 27: 1256-1262.

[18] HaasBJ, SalzbergSL, ZhuW, PerteaM, AllenJE, OrvisJ, WhiteO, BuellCR, WortmanJR. Automated eukaryotic gene structure annotation using EVidenceModeler and the Program to Assemble Spliced Alignments. Genom Biol, 2008, 9: 1-22.

[19] HanC, ZhuQ, LuH, WangC, ZhouX, PengC, TangL, HanL, ChenJ, LiS, et al. . Screening and characterization of sex-specific markers developed by a simple NGS method in mandarin fish (Siniperca chuatsi). Aquaculture, 2020, 527: 735495.

[20] HanC, WangC, OuyangH, ZhuQ, HuangJ, HanL, LiS, LiG, LinH, ZhangY. Characterization of dmrts and their potential role in gonadal development of mandarin fish (Siniperca chuatsi). Aqua Rep, 2021, 21: 100802.

[21] HattoriRS, MuraiY, OuraM, MasudaS, MajhiSK, SakamotoT, FernandinoJL, SomozaGM, YokotaM, StrüssmannCA. A Y-linked anti-Mullerian hormone duplication takes over a critical role in sex determination. Proc Natl Acad Sci USA, 2012, 109: 2955-2959.

[22] HattoriR, KumazawaK, NakamotoM, NakanoY, YamaguchiT, KitanoT, YamamotoE, FujiK, SakamotoT. Y-specific amh allele, Amhy, is the master sex-determining gene in Japanese flounder Paralichthys olivaceus. Front Genet, 2022, 13: 1007548.

[23] HeS, LiL, LvLY, CaiWJ, DouYQ, LiJ, TangSL, ChenX, ZhangZ, XuJ, et al. . Mandarin fish (Sinipercidae) genomes provide insights into innate predatory feeding. Commun Biol, 2020, 3: 361.

[24] HulataG, WohlfarthG, RothbardS. Progeny-testing selection of tilapia broodstocks producing all-male hybrid progenies-preliminary results. Aquaculture, 1983, 33: 263-268.

[25] KarkiR, ManSM, MalireddiRS, KesavardhanaS, ZhuQ, BurtonAR, SharmaBR, QiX, PelletierX, VogelP, et al. . NLRC3 is an inhibitory sensor of PI3K-mTOR pathways in cancer. Nature, 2016, 540: 583-587.

[26] KorenS, WalenzBP, BerlinK, MillerJR, BergmanNH, PhillippyAM. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genom Res, 2017, 27: 722-736.

[27] LiXY, GuiJF. Diverse and variable sex determination mechanisms in vertebrates. Sci China Life Sci, 2018, 61: 1503-1514.

[28] LiM, YangH, ZhaoJ, FangL, ShiH, LiM, SunY, ZhangX, JiangD, ZhouL, et al. . Efficient and Heritable Gene Targeting in Tilapia by CRISPR/Cas9. Genetics, 2014, 197: 591-599.

[29] LiM, SunY, ZhaoJ, ShiH, ZengS, YeK, JiangD, ZhouL, SunL, TaoW, et al. . A tandem duplicate of anti-Mullerian hormone with a missense SNP on the Y chromosome is essential for male sex determination in Nile Tilapia, Oreochromis Niloticus. Plos Genet, 2015, 11: e1005678.

[30] LiM, ZhangR, FanG, XuW, ZhouQ, WangL, LiW, PangZ, YuM, LiuQ, et al. . Reconstruction of the origin of a neo-Y sex chromosome and its evolution in the spotted knifejaw, Oplegnathus punctatus. Mol Biol Evol, 2021, 38: 2615-2626.

[31] LiXY, MeiJ, GeCT, LiuXL, GuiJF. Sex determination mechanisms and sex control approaches in aquaculture animals. Sci China Life Sci, 2022, 65: 1091-1122.

[32] LiX, BieL, WangY, HongY, ZhouZ, FanY, YanX, TaoY, HuangC, ZhangY, et al. . LINE-1 transcription activates long-range gene expression. Nat Genet, 2024, 56: 1494-1502.

[33] LichilínN, El TaherA, BöhneA. Sex-biased gene expression and recent sex chromosome turnover. Philos Transact Roy Soc B-Biol Sci, 2021, 376: 20200107.

[34] LiuX, DaiS, WuJ, WeiX, ZhouX, ChenM, TanD, PuD, LiM, WangD, et al. . Roles of anti-Müllerian hormone (Amh) and its duplicates in sex determination and germ cell proliferation of Nile tilapia. Genetics, 2021, 220: iyab237.

[35] Liu S, Han C, Huang J, Li M, Yang J, Li G, Lin H, Li S, Zhang Y. Genome-wide identification, evolution and expression of TGF-β signaling pathway members in mandarin fish (Siniperca chuatsi). Internat J Biol Macrom. 2023a;31:126949. https://doi.org/10.1016/j.ijbiomac.2023.126949.

[36] Liu S, Han C, Zhang Y. De novo assembly, characterization and comparative transcriptome analysis of gonads reveals sex-biased genes in Coreoperca whiteheadi. Comp Biochem Physiol Part D. 2023b;1:101115. https://doi.org/10.1016/j.cbd.2023.101115.

[37] MeiselRP. Evolution of sex determination and sex chromosomes: a novel alternative paradigm. BioEssays, 2020, 42: 1900212.

[38] MelamedE, ArnoldA. The role of LINEs and CpG islands in dosage compensation on the chicken Z chromosome. Chrom Res, 2009, 17: 727-736.

[39] MengS, LiuX, ZhuS, XieP, FangH, PanQ, FangK, LiF, ZhangJ, CheZ, et al. . Young LINE-1 transposon 5’UTRs marked by elongation factor ELL3 function as enhancers to regulate naïve pluripotency in embryonic stem cells. Nat Cell Biol, 2023, 25: 1319-1331.

[40] MyoshoT, OtakeH, MasuyamaH, MatsudaM, KurokiY, FujiyamaA, NaruseK, HamaguchiS, SakaizumiM. Tracing the emergence of a novel sex determining gene in medaka, Oryzias luzonensis. Genetics, 2012, 191: 163-170.

[41] Pan Q, Feron R, Yano A, Guyomard R, Jouanno E, Vigouroux E, Wen M, Busnel JM, Bobe J, Concordet JP, et al. Identification of the master sex determining gene in Northern pike (Esox lucius) reveals restricted sex chromosome differentiation. PLoS Genet. 2019;15:e1008013. https://doi.org/10.1371/journal.pgen.1008013.

[42] PeichelCL, McCannSR, RossJA, NaftalyAFS, UrtonJR, CechJN, GrimwoodJ, SchmutzJ, MyersRM, KingsleyDM, et al. . Assembly of the threespine stickleback Y chromosome reveals convergent signatures of sex chromosome evolution. Genom Biol, 2020, 21: 177.

[43] Ross JA, Urton JR, Boland J, Shapiro MD, Peichel CL. Turnover of sex chromosomes in the stickleback fishes (Gasterosteidae). PLoS Genet. 2009;5:e1000391. https://doi.org/10.1371/journal.pgen.1000391.

[44] SacchiS, D’IppolitoG, SenaP, MarsellaT, TagliasacchiD, MaggiE, ArgentoC, TirelliA, GiuliniS, MarcaAL, et al. . The anti-Müllerian hormone (AMH) acts as a gatekeeper of ovarian steroidogenesis inhibiting the granulosa cell response to both FSH and LH. J Ass Reprod Genet, 2016, 33: 95-100.

[45] SchartlM, SchmidM, NandaI. Dynamics of vertebrate sex chromosome evolution: from equal size to giants and dwarfs. Chromosoma, 2016, 125: 553-571.

[46] Shearwin-Whyatt L, Fenelon J, Yu H, Major A, Qu Z, Zhou Y, Shearwin KE, Galbraith J, Stuart A, Adelson D, et al. AMHY and sex determination in egg-laying mammals. bioRxiv. 2024. https://doi.org/10.1101/2024.04.18.588952.

[47] SkinnerBM, SargentCA, ChurcherC, HuntT, HerreroJ, LovelandJE, DunnM, LouzadaS, FuB, ChowW, et al. . The pig X and Y chromosomes: structure, sequence, and evolution. Genome Res, 2015, 26: 130-139.

[48] SohYQS, AlföldiJ, PyntikovaT, BrownLG, GravesT, MinxPJ, FultonRS, KremitzkiC, KoutsevaN, MuellerJL, et al. . Sequencing the mouse Y chromosome reveals convergent gene acquisition and amplification on both sex chromosomes. Cell, 2014, 159: 800-813.

[49] SunC, NiuY, YeX, DongJ, HuW, ZengQ, ChenZ, TianY, ZhangJ, LuM, et al. . Construction of a high-density linkage map and mapping of sex determination and growth-related loci in the mandarin fish (Siniperca chuatsi). BMC Genom, 2017, 18: 446.

[50] TakehanaY, NaruseK, HamaguchiS, SakaizumiM. Evolution of ZZ/ZW and XX/XY sex-determination systems in the closely-related medaka species Oryzias hubbsi and O. dancena. Chromosoma, 2007, 116: 463-470.

[51] TakehanaY, MatsudaM, MyoshoT, SusterML, KawakamiK, Shin-IT, KoharaY, KurokiY, ToyodaA, FujiyamaA, et al. . Co-option of Sox3 as the male-determining factor on the Y chromosome in the fish Oryzias dancena. Nat Commun, 2014, 5: 4157.

[52] Tao W, Yuan J, Zhou L, Sun L, Sun Y, Yang S, Li M, Zeng S, Huang B, Wang D. Characterization of gonadal transcriptomes from Nile tilapia (Oreochromis niloticus) reveals differentially expressed genes. PLoS ONE. 2013;8: e63604. https://doi.org/10.1371/journal.pone.0063604.

[53] TaoW, XuL, ZhaoL, ZhuZ, WuX, MinQ, WangD, ZhouQ. High-quality chromosome-level genomes of two tilapia species reveal their evolution of repeat sequences and sex chromosomes. Mol Ecol Res, 2021, 21: 543-560.

[54] VicosoB. Molecular and evolutionary dynamics of animal sex-chromosome turnover. Nat Ecol Evol, 2019, 3: 1632-1641.

[55] Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, Wortman J, Young SK, et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS ONE. 2014;9:e112963. https://doi.org/10.1371/journal.pone.0112963.

[56] WenM, ZhangY, WangS, WangY, QinQ, TaoM, ZhangC, ZhaoR, WangS, HuF, et al. . Characterization of sex locus and sex-specific sequences in the mandarin fishes. Aquaculture, 2022, 561: 738650.

[57] WrightAE, DeanR, ZimmerF, MankJE. How to make a sex chromosome. Nat Commun, 2016, 7: 12087.

[58] XuL, AuerG, PeonaV, DengY, FengS, ZhangG, BlomMPK, ChristidisL, ProstS, IrestedtM, et al. . Dynamic evolutionary history and gene content of sex chromosomes across diverse songbirds. Nat Ecol Evol, 2019, 3: 834-844.

[59] XueL, GaoY, WuM, TianT, FanH, HuangY, HuangZ, LiD, XuL. Telomere-to-telomere assembly of a fish Y chromosome reveals the origin of a young sex chromosome pair. Genom Biol, 2021, 22: 1-20.

[60] YangC, ChenL, HuangR, GuiB, LiY, LiY, LiY, LiaoL, ZhuZ, WangY, et al. . Screening of genes related to sex determination and differentiation in mandarin fish (Siniperca chuatsi). Int J Mol Sci, 2022, 23: 7692.

[61] YiS, CharlesworthB. Contrasting patterns of molecular evolution of the genes on the new and old sex chromosomes of Drosophila miranda. Mol Biol Evol, 2000, 17: 703-717.

[62] ZhangW, WangX, YuQ, MingR, JiangJ. DNA methylation and heterochromatinization in the male-specific region of the primitive Y chromosome of papaya. Genom Res, 2008, 18: 1938-1943.

[63] Zhu Q, Han C, Liu S, Ouyang H, Liu D, Zhang Z, Huang J, Han L, Li S, Li G, et al. Development and gene expression analysis of gonad during 17α-methyltestosterone-induced sex reversal in mandarin fish (Siniperca chuatsi). Aqua Rep. 2022;23:101049. https://doi.org/10.1016/j.aqrep.2022.101049.

Funding

National Key R&D Program of China(2024YFD2401500)

National Natural Science Foundation of China(32302995)

the Guangdong Basic and Applied Basic Research Foundation(2022A1515110140)

the Science and Technology Planning Project of Guizhou(2023-085)

the Science and Technology Planning Project of Guangzhou(2025D04J0094)

Guangzhou Municipal Joint Funding Project of Universities (Institutes) and Enterprises(2025A03J0022)

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